Clustering and Maintenance Mechanisms of Kaposi's Sarcoma-associated Herpesvirus in Tumor Cell

Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic virus associated with primary effusion lymphoma (PEL), Kaposi’s sarcoma (KS), and multicentric Castleman’s disease. It is known that these KSHV-associated tumors rely on the KSHV genome to maintain their viability; in the absence of viral genomes, these tumor cells do not survive. Therefore, understanding the mechanisms by which KSHV genomes become established and stably maintained in tumor cells is important to gain insight into the development and progression of such tumors, and can be crucial to the discovery of novel therapeutic approaches for eliminating KSHV-associated tumors. It is known that replication of KSHV genomes during viral latency depends on its latent origin of replication located in multiple copies of terminal repeats (TR), as well as a viral protein, LANA1. a minimum of 16 copies seems to be required for KSHV to sustain tumor cells. My previous research revealed that the number of KSHV genomes is distributed in a wide range among a tumor population, yet no cell has zero KSHV genomes. Interestingly, a similar distribution of KSHV genomes could be recapitulated from a single cell, showing that KSHV genomes likely partition unequally to daughter cells during cell division. The absence of cells with non-zero viral genomes also reflects selective advantages that are provided by KSHV. Additionally, the computational simulations, which were developed on the basis of measurements from FISH to predict the distributions of KSHV genomes in cells observed with live-cell imaging, suggest that clustering of KSHV genomes facilitates not only their unequal partitioning but also their establishment in cells early after infection. It has been shown that LANA1 binds to the LANA1-binding sites (LBS) on TRs (of which each contains at least two LBSs) as a dimer through its C-terminus, to support the synthesis of KSHV genomes. LANA1 tethers KSHV genomes to chromosomes through the interaction of histone H2A/H2B with its N-terminus, and this is capable of inducing a 30-nm chromatin fiber structure. The spacing between the LBSs of two TRs yields a spatial configuration in which each TR wraps around a set of 4 histone octamers, and this spatial arrangement is also required to support the establishment of KSHV genomes in cells. This study hypothesizes that the spatial arrangement of the LBSs on TRs leads to the N-terminus of two LANA1 dimers to face outward in the same orientation for each 12 nucleosomes embedded in the 30-nm chromatin fiber of the KSHV genome. Accordingly, 10-12 dimers of LANA1 on a KSHV genome are capable of associating with nucleosomes on sister DNA molecules by “zippering” them together. This study proposes to test these hypotheses and further investigate the mechanisms underlying KHSV genome clustering, to understand how clustering contributes to the distribution of KSHV genomes in host cells. Moreover, this study will elucidate the tethering mechanisms of KSHV genomes to host chromosomes, and assess how these processes affect viral establishment and stable maintenance in newly infected cells. The findings proposed in this study are expected to yield novel insights into how the KSHV genome propagates and drives oncogenesis, while also providing a foundation for the potential development of novel anti-cancer strategies that seek to eradicate KSHV genomes from tumor cells, thereby eliminating oncogenic potential or the tumor cells themselves to provide disease relief.

Project ID:PG10701-0050
External Project ID:NHRI-EX107-10623BI